Stimulation of median raphe terminals in dorsal CA2 reduces social investigation in male mice specifically investigating social stimulus of ovariectomized female mice

The cornu ammonis area 2 (CA2) region is essential for social behaviors, especially in social aggression and social memory. Recently, we showed that targeted CA2 stimulation of vasopressin presynaptic fibers from the paraventricular nuclei of hypothalamus strongly enhances social memory in mice. In addition, the CA2 area of the mouse hippocampus receives neuronal inputs from other regions including the septal nuclei, the diagonal bands of Broca, supramammillary nuclei, and median raphe nucleus. However, the functions of these projections have been scarcely investigated. A functional role of median raphe (MR) – CA2 projection is supported by the MR to CA2 projections and 82% reduction of hippocampal serotonin (5-HT) levels following MR lesions. Thus, we investigated the behavioral role of presynaptic fibers from the median raphe nucleus projecting to the dorsal CA2 (dCA2). Here, we demonstrate the optogenetic stimulation of 5-HT projections to dCA2 from the MR do not alter social memory, but instead reduce social interaction. We show that optical stimulation of MR fibers excites interneurons in the stratum radiatum (SR) and stratum lacunosum moleculare (SLM) of CA2 region. Consistent with these observations, we show that bath application of 5-HT increases spontaneous GABA release onto CA2 pyramidal neurons and excites presumed interneurons located in the SR/SLM. This is the first study, to our knowledge, which investigates the direct effect of 5-HT release from terminals onto dCA2 neurons on social behaviors. This highlights the different roles for these inputs (i.e., vasopressin inputs regulating social memory versus serotonin inputs regulating social interaction).


Subjects
Stimulus mice used in social recognition testing were sexually naïve, ovariectomized Balb/c adult females.They were purchased from Jackson Labs (Stock # 000651) and ovariectomized.Briefly, a small dorsal midline incision was made, the muscle wall spread using forceps, and the ovaries were removed.Following a two-week recovery period, females were singly housed for at least one week prior to testing.

Raphe-cre
Mice were genotyped by PCR using DNA extracted from tail snips.Raphe-cre mice were genotyped using a forward primer, Cre.m35 (AGAACCTGAAGATGTTCGCGATTATCTTCTATATC) and reverse primer, Cre.c33 (TAGTTACCCCCAGGCTAAGTGCCTTCTCTACAC) with PCR product of 460 bp.PCR was carried out for 40 cycles with denaturation at 95°C, annealing at 65°C, and extension at 72°C, all for 45s.

Stereotaxic surgery, viral tracing, and optogenetic surgical implantation
The recombinant adeno-associated virus (AAV) vectors were serotyped with AAV2 coat proteins and packaged by the University of North Carolina Vector Core (Chapel Hill, NC, USA) or Addgene (MA, USA).Male mice (10-12 weeks old) were anesthetized with 100 mg/kg ketamine (100 mg/mL) and 50 mg/kg xylazine (20 mg/mL) and placed on a stereotaxic apparatus.The head was leveled and a small incision was made to expose the skull for localization of the reference lambda and bregma sutures.Burr holes were drilled for subsequent viral injections or optic fiber placements and dental cement was used to hold the optic fiber placements.

Histology and immunohistochemistry
Brains were processed with immunohistochemistry to detect cells expressing 5-HT and GFP in tissues.Mice were deeply anesthetized and transcardially perfused with 0.9 % saline with 10 U/mL of heparin (heparin lithium salt, Cat.#H-0878, Sigma, St Louis, MO, USA), followed by 4%

Behavioral Analysis
Videos of behavioral tests were recorded from above and coded by an observer blind to the identity of the mouse using Noldus Observer v12.The total duration and frequency of behavior is reported.
The behavioral tests were conducted in a week apart from each test.

Social recognition (SRM) test
Duration of sniffing was manually scored, and experimenter was blinded to the subject.Sniffing was only recorded when the experimental mouse initiated the behavior.When that mouse contacted the stimulus mouse with its nose, a sniffing bout was initiated and when nose of experimental mouse was no longer in contact, the bout was ended.

Object recognition
Duration of sniffing was manually scored, and experimenter was blinded to the subject.When experimental mouse contacted the object with their nose, a sniffing bout was initiated and when nose of experimental mouse was no longer in contact, the bout was ended.

Social interaction test
Duration of sniffing was manually scored, and experimenter was blinded to the subject.When experimental mouse contacted the stimulus mouse or object with their nose, a sniffing bout was initiated and when nose of experimental mouse was no longer in contact, the bout was ended.
Chamber and objects were cleaned thoroughly with 70% ethanol between trials.

Marble Burying Test
Latency to begin digging around the first marble was manually scored, and experimenter was blinded to the subject.

Open-field test
For locomotor activity, distance travelled was measured.Center was defined on Ethovision as the inner 50% zone of the open-field arena, center duration as the time spent in the inner zone, and center frequency as the frequency to enter inner zone. .There were no significant differences between the genotypes and stimulations.

Supplemental Figure 2 .Supplemental Figure 3 .
We also observed 1 cell in the SR, proximal to CA2, which showed a membrane hyperpolarization in response 10 light pulses delivered at 20 Hz. 9 Object recognition memory test with 2-hour interval.(A) Illustrates Raphe-cre mice optogenetically stimulated during acquisition period (n = 8).(B) Non-stimulated Raphe-cre group (n = 9).(C) WT mice optogenetically stimulated during acquisition period (n = 8).(D) Object sniffing duration during acquisition and retrieval period.(E) Change in sniffing